FANCD2 and RAD51 Regulate PCNA Monoubiquitination and Translesion Synthesis

FANCD2 is a key player in Fanconi anemia (FA) pathway. It has been shown that PCNA and Rad18 can interact with FANCD2 and regulate FANCD2 monoubiquitination. Rad18 is the E3 ubiquitin ligase responsible for PCNA monoubiquitination, which is critical for PCNA function in translesion synthesis (TLS). Previous work by us and others has shown that the FA pathway interacts with PCNA and RAD18. Even though FA cells demonstrate unequivocal sensitivity to crosslinkers such as mitomycin C (MMC), we find surprisingly that they are largely resistant to hydroxyurea (HU), except for cells containing no expression of FANCD2. In order to understand the mechanism for this finding, we explored the interaction of FANCD2, Rad51, Rad18, and PCNA, which form a complex and is enhanced upon HU exposure. While FANCD2 is required, monoubiquitination of FANCD2 is not. In addition, PCNA monoubiqutination follows exactly the same pattern. The monoubiquitination of PCNA in response to HU in FANCA deficient mutant confirms that non-ubiquitinated FANCD2 can still support PCNA monoubiquitination. Rad51, but not BRCA1 is also required in regulating PCNA monoubiquitination in response to HU, suggesting that this function is independent of homologous recombination (HR). We further show that translesion polymerase Pol H chromatin localization is decreased in FANCD2 deficient cells, FANCD2 siRNA knock down cells and Rad51 siRNA knock down cells. Our data suggest that FANCD2 and Rad51 play an important role in PCNA monoubiquitination and TLS in a FANCD2 monoubiquitination and HR-independent manner in response to HU. This effect is not observed with mitomycin C (MMC) treatment, indicating that DNA repair systems differentially respond to different types of DNA damage.